The present invention relates to contrast agents for diagnostic imaging. In particular, this invention relates to novel multimeric compounds which exhibit improved affinity for physiologically relevant targets, such as proteins, and surprisingly improved relaxivity properties upon binding. The compounds comprise:
a) two or more Image Enhancing Moieties (xe2x80x9cIEMsxe2x80x9d)
b) two or more Target Binding Moieties (xe2x80x9cTBMsxe2x80x9d), providing for in vivo localization and multimer rigidification;
c) a scaffold framework for attachment of the above moieties (xe2x80x9cscaffoldxe2x80x9d);
d) optional linkers for attachment of IEMs to the scaffold (xe2x80x9clinkerxe2x80x9d).
This invention also relates to pharmaceutical compositions comprising these compounds and to methods of using the compounds and compositions for contrast enhancement during imaging.
Diagnostic imaging techniques, such as magnetic resonance imaging (MRI), X-ray, nuclear radiopharmaceutical imaging, ultraviolet-visible-infrared light imaging, and ultrasound, have been used in medical diagnosis for a number of years. Contrast media additionally have been used to improve or increase the resolution of the image or to provide specific diagnostic information. In some cases, such as imaging with ultrasound, the introduction of contrast media has been recent.
To be effective, the contrast media must interfere with the wavelength of electromagnetic radiation used in the imaging technique, alter the physical properties of tissue to yield an altered signal, or, as in the case of radiopharmaceuticals, provide the source of radiation itself. MRI and optical imaging methods are unique among imaging modalities in that they yield complex signals that are sensitive to the chemical environment. While the signal from X-ray or radionuclide agents remains the same whether the agents are free in plasma, bound to proteins or other targets, or trapped inside bone, certain contrast agents for MRI and optical imaging will have different signal characteristics in differing physiological environments. An optical dye may exhibit changes in its absorbance, reflectance, fluorescence, phosphorescence, chemiluminescence, scattering, or other spectral properties upon binding. It is important that the contrast agent be sufficiently sensitive and present at high enough concentration so that signal changes can be observed.
Attempts to Improve Contrast by Increasing the Number of IEMs
Targeted agents should deliver meaningful concentrations of the imaging moiety to the target so that sufficient improvement in the signal is observed during the course of imaging. Achieving sufficient sensitivity is a significant problem for MRI in particular, where concentrations in the range of 10-1000 micromolar (xcexcM) of the image enhancing moiety are required to produce an adequate signal. The problem can be further complicated for targeted agents if the desired target is present at low concentrations. For example, in order to image biological receptor targets that are present at less than xcexcM concentrations, greater signal enhancement is required at the target site to provide sufficient image contrast. Increased contrast has been approached by using (1) drug delivery vehicles to provide high local concentrations of the contrast agent, (2) multiple IEMs in a single contrast agent, [see, for example, Martin V. V., et al., Bioconjug. Chem., 6: pp. 616-23 (1995); Shukla, R. et al., Mag. Reson. Med., 35: pp. 928-931 (1996); Ranganathan, R. S., et al., Invest. Radiol., 33: pp. 779-797 (1998)], or (3) particular IEMs of defined structure with improved signal enhancement properties. The ideal targeted contrast agent should efficiently combine IEMs and improved signal enhancement properties.
To incorporate a high number of image enhancing moieties into a contrast agent, large concentrations of low molecular weight contrast agents have been packaged within suitable drug delivery vehicles, such as polymerized vehicles or liposomes [Bulte J. W., et al., J. Magn. Reson. Imaging, 9: pp. 329-335 (1999)]. Unfortunately, these materials are difficult to direct to a target.
To increase the number of the image enhancing moieties, investigators have, for example, created polymers, dendrimers, and organic compounds in association with multiple IEMs. High numbers of IEMs, such as Gd(III) chelates for MRI, can be covalently attached to polymers [Schuhmann-Giampieri, G. et al. J. Invest. Rad., 26: pp. 969-974 (1991); Corot, C. et al. Acta Rad., 38:S412 pp. 91-99 (1997)] and dendrimers [Jacques, V., et al., J. Alloys Cmpd., 249: pp. 173-177 (1997); Margerum, L. D., et al., J. Alloys Compd., 249: pp. 185-190 (1997); Toth, E., et al., Chem. Eur. J., 2: pp. 1607-1615 (1996)]. Polymeric agents typically comprise a mixture of species with a broad and complex molecular weight distribution. These heterogeneous properties adversely affect agent performance and make characterization difficult. Furthermore, it is synthetically difficult to selectively introduce TBMs along with multiple IEMs. Therefore there exists a need for well-defined, homogeneous molecules for use as contrast agents that can provide adequate image enhancement at a target.
Dendrimers (such as xe2x80x9cStarburst dendrimersxe2x80x9d, or xe2x80x9ccascade polymersxe2x80x9d) theoretically offer a single high molecular weight species onto which many IEMs can be covalently attached. [Fischer, M. et al. Angew. Chem., Int. Ed. Eng., 38/7: pp. 884-905 (1999); Weiner, E. C. et al., Mag. Reson. Med., 31: pp. 1-8 (1994)]. However, dendrimers, like polymeric agents, present significant synthetic problems, especially when selectively introducing tissue-specific targeting groups.
Organic molecules have been synthesized with multiple image enhancing moieties. MRI contrast agents of this type are referred to herein as xe2x80x9cmultimeric chelatesxe2x80x9d or xe2x80x9cmultimersxe2x80x9d and typically comprise 2-12 IEMs. [Shukla, R. et al., Mag. Reson. Med., 35: pp. 928-931 (1996); Shukla, R. B., et al., Acta Radiol., 412: pp. 121-123 (1997); Ranganathan, R. S., et al., Invest. Radiol., 33: pp. 779-797 (1998)]. Advantages of multimeric chelates include: (1) they are homogeneous molecules in that they have a single size and structure, unlike polymers and dendrimers, (2) they can be readily synthesized and purified, and (3) targeting groups can be readily incorporated. Unfortunately, the ability of multimeric chelates to improve the MRI signal intensity has been disappointingly low. This is because the proton relaxation rate enhancement (or xe2x80x9crelaxivityxe2x80x9d), which correlates with signal enhancement, has decreased as the number of IEMs was increased. Therefore, contrast agents wherein the relaxivity does not decrease when the number of IEMs increases are needed to achieve greater signal enhancement at a target.
Attempts to Improve Contrast by Decreasing the Rotational of the Contrast Agent
Attempts have been made to increase the relaxivity of non-targeted multimeric MRI contrast agents by restricting rotational motion. Attempts to restrict rotational motion have focused on (1) decreasing the flexibility of the molecule or (2) restricting rotational motion through binding to a target.
For example, non-targeted agents have been synthesized with rigid frameworks to which multiple Gd(III) chelates are attached [Shukla, R. et al., Mag. Reson. Med., 35: pp. 928-931 (1996); Shukla, R. B., et al., Acta Radiol., 412: pp. 121-123 (1997); Ranganathan, R. S., et al., Invest. Radiol., 33: pp. 779-797 (1998); Jacques, V., et al., J. Alloys Cmpd., 249: pp. 173-177 (1997)]. However, these structures have several drawbacks. First, the relaxivities per Gd(III) ion that have been achieved for agents containing more than two chelates has been less than that observed for single chelates, such as MS-325. Therefore, local chelate motion could still be further reduced. Second, these agent are not targeted. More importantly, even if they were targeted, rigid multimer frameworks would greatly increase the unwanted background signal because the signal enhancement is significant regardless of whether the contrast agent is bound to a target or not. Therefore, there exists a need for contrast agents that enhance an image of a target only when bound to the target.
Rotational motion of a single IEM can be effectively limited upon non-covalent target binding, resulting in a relaxivity increase for the target-bound forms of as much as 5-10 fold [U.S. Pat. No. 4,880,008]. This relaxivity increase is as good as or better than that observed for IEMs that are covalently linked to the target [Schmiedl, U., Ogan, M., Paajanen, H., Marotti, M., Crooks, L. E., Brito, A. C., and Brasch, R. C. Radiology (1987) 162: pp.205-210; Ogan, M. D., Schmiedl, U., Moseley, M. E., Grodd, W., Paajanen, H., and Brasch, R. C. Invest. Radiol. (1987) 22: pp. 665-71]. Examples of agents which exploit this effect are the liver protein-targeted contrast agents Gd-EOB DTPA [Runge V. M. Crit. Rev. Diagn. Imaging 38: pp. 207-30 (1997)] and Gd-BOPTA [Kirchin M. A., et al., Invest. Radiol., 33: pp. 798-809 (1998)] or the albumin-targeted agents MS-325 [Lauffer, R. B., et al., Radiology, 207: pp. 529-538 (1998)] and MP-2269 [Hofman Mark B. M. et al. Acadademic Radiology, 5(suppl 1): S206-S209 (1998)]. Relaxivity increases of approximately 7-fold were reported for MS-325 (47 mMxe2x88x921sxe2x88x921) as a result of non-covalent binding to serum albumin [Lauffer, R. B., et al., Radiology, 207: pp. 529-538 (1998)]. 
Upon binding to albumin, the monomeric contrast agent MS-325, having the chemical structure shown by Formula (I), exhibits an increase in signal enhancement. When bound, the complex tumbles at a slower rate than in the unbound state which results in greater relaxivity. Surprisingly, however, the addition of multiple IEMs to targeted contrast agents, such as MS-325, has failed to enhance contrast further because relaxivity decreases at the individual gadolinium centers in the multimeric structure. For example, an albumin-targeted multimer with four Gd(III) ions exhibited molecular relaxivities per Gd(III) of only 9-13 mmxe2x88x921sxe2x88x921 compared to a relaxivity of 47 mMxe2x88x921sxe2x88x921 for MS-325, which contains a single Gd(III) [Martin V. V., et al., Bioconjug. Chem., 6: pp. 616-23 (1995)]. Thus, the relaxivity of a targeted multimeric chelate is typically much less per Gd (III) than that observed for the analogous targeted single chelate.
Rationale
Table 1 demonstrates that merely increasing the number of IEMs is insufficient to improve total relaxivity because the relaxivity per IEM decreases as the number of IEMs increases despite the presence of the target binding group comprising two phenyl rings. To understand Table 1, it is important to define the extent to which a target-binding MRI contrast agent can achieve its maximum possible relaxivity. This maximum relaxivity for a particular contrast agent is approximately equal to the relaxivity of the molecule when bound to a target (R1bound), such as Human Serum Albumin (HSA). The average bound is a normalized measure of the average relaxivity for all bound species under a standard set of conditions (such as a specific target or protein concentration, drug concentration, temperature, etc.) that is weighted by the bound population of each species. Therefore, since the value of bound is a normalized quantity, comparisons of relaxivities can be made among different molecules in the bound state by comparing values for bound. Comparison of the calculated bound values provides a convenient method for comparing compounds irrespective of their affinities for a target.
Calculating the average (bound) requires measuring the relaxivity of the free chelate (R1free) as well as the observed relaxivity (R1obs) and percent binding of the agent to a target solution typically containing 4.5% of the target, e.g., HSA. The R1obs is a mole fraction (x) weighted average of R1free and R1bound:             R1      obs        =                            x          free                ⁢                  R1          free                    +                        ∑          1                ⁢                  xe2x80x83                ⁢                              x            i                    ⁢                      R1                          bound              ,              i                                                              where        ⁢                  xe2x80x83                ⁢                  x          free                    +                        ∑          1                ⁢                  xe2x80x83                ⁢                  x          i                      =          1      ⁢              xe2x80x83            ⁢      and                          ∑        1            ⁢              xe2x80x83            ⁢              x        i              =          x      bound      
Thus:       ℝ𝕀    bound    =                    R1        tts            -                        x          free                ⁢                  R1          free                            x      bound      
The chemical structures and bound relaxivities of a series of albumin-targeted contrast agents are shown in Table 1. In this set of compounds, a compound with a single IEM (i.e., MS-325) is compared with a series of multimers comprising multiple IEMs, but the same diphenylcyclohexyl albumin TBM and the methylene phosphate group are present in all compounds.
FIG. 1 is a graphical representation of the same data shown in Table 1. The average bound per IEM, in this case a Gd(III) chelate, at 20 MHz is plotted against the number of IEMs for a series of multimeric contrast agents containing the a single diphenylcyclohexyl protein binding group.
In the molecules of Table 1 and FIG. 1, both the structure of the gadolinium chelate (IEM), the methylene phosphate group, and the diphenylcyclohexyl group (TBM) remain constant. The data in Table 1 and FIG. 1 show that as the number of chelated paramagnetic metal ions increases, the relaxivity per metal ion is reduced. The number of Gd(III) chelates varies from one (MS-325) to four, but despite this four-fold increase in the number of IEMs, the total relaxivity increases by only about 50%. This modest increase in total relaxivity is a consequence of the decreasing relaxivity per Gd(III) ion. Note that the average bound per Gd(III) decreases from 47 mMxe2x88x921 sxe2x88x921 to 14.9 mMxe2x88x921sxe2x88x921 despite the contrast agent being bound. This decrease is due to local chelate motion which surprisingly increases with the number of IEMs despite multiple aromatic rings in the single TBM.
Apparently, increasing the number of chelating moieties also increases the rotational freedom of the molecule, at least near the sites of gadolinium chelation. The decrease in relaxivity is especially notable as the size increases beyond two chelated gadolinium ions per multimer molecule. For example, in the case of M8-03 the total relaxivity per gadolinium is only about 15 mMxe2x88x921sxe2x88x921, approximately one third that observed for MS-325. The total relaxivity for the compound M8-03 is therefore only 60 mMxe2x88x921sxe2x88x921, just 1.3 times that of MS-325 although four times as many IEMs are present. Obviously, such a modest increase in relaxivity does not justify the added synthetic complexity and cost to develop such agents for in vivo MR imaging. Thus, the simple combination of multiple image enhancing moieties with a single target binding moiety does not generate a commensurate increase in relaxivity. Thus, there exists a need to synthesize multimeric MRI contrast agents wherein the relaxivity at each chelate is maintained even as the number of IEMs increases.
Overall, immobilization of a target-bound contrast agent can be remarkably effective at increasing the relaxivity for a single chelate (e.g. MS-325) but is rather ineffective for multimeric chelates. That is, in order to increase the relaxivity at each chelate site, it is necessary to both reduce the overall rotational correlation time for the molecule and to reduce the local chelate motion at each chelation site. There remains a need for a mechanism to efficiently immobilize target-binding multimeric contrast agents so that more effective signal enhancement is produced during imaging.
A method is needed to improve signal contrast at specific targets. The problem has been approached by (1) increasing the number of IEMs, or (2) decreasing the flexibility of the molecule. Increasing the number of IEMs has been unsuccessful because the contrast agents are not of homogeneous size and structure, pose synthetic difficulties, are difficult to target, or fail to increase contrast proportionately with the increase in IEM number. Decreasing the flexibility has been unsuccessful because rigid contrast agents create high background when unbound. Binding of a multimer to a target through a single TBM is not sufficient to both decrease flexibility and increase relaxivity significantly. Therefore, a need exists to improve contrast at specific targets by increasing the number of IEMs while simultaneously decreasing the flexibility of the molecule only when bound to the target.
The current invention provides a mechanism to greatly improve the efficacy of in vivo contrast agents. Great improvements in contrast (signal to noise) at the target are possible if multimeric contrast agents are flexible in the unbound state (resulting in low relaxivity and a weak signal) and less flexible in the bound state (resulting in high relaxivity and a strong signal). That is, it is more important to rigidify the multimeric contrast agent in the bound state than in the unbound state since this minimizes background in the unbound state while high relaxivity is maintained in the bound state. Such agents are bound to proteins or other specific targets by non-covalent interactions at two or more separate loci. Multilocus binding is achieved by incorporating two or more TBMs into the agent, each of which has some affinity for one or more sites on the target.
More specifically, the invention relates to the use of xe2x80x9cmultilocus,xe2x80x9d non-covalent interactions between a contrast agent with multiple IEMs (a xe2x80x9cmultimerxe2x80x9d) and a target to simultaneously 1) induce binding to the target (thus giving specificity), 2) anchor several IEMs to the target and 3) thereby rigidify the multiple IEM structure. A key aspect of the invention is that the contrast agent is less flexible in the bound state than in the unbound state. Binding of the contrast agent to the target increases the relaxivity and signal intensity of a metal chelate IEM by increasing the overall rotational correlation time of the metal ionxe2x80x94imaging atom vector, i.e., by limiting rotational motion. Multilocus binding enables further relaxivity enhancement by decreasing the flexibility of the multiple chelate structure in the bound state both in general and at the local sites where chelate motion occurs. The flexibility of the molecule in the unbound state provides particular advantages over previously described multimeric MRI agents with rigid structures linking the chelates and with no difference in rigidity between a bound and unbound state. [Ranganathan, R. S., et al., Invest. Radiol., 33: pp. 779-797 (1998)]. The multilocus binding concept for multimeric chelates is shown schematically in FIG. 2. Specifically, FIG. 2 shows the key components of an example multimeric contrast agent bound to a target through multilocus interactions. Three important features of the contrast agent illustrated by the drawing are: (1) multiple separate TBMs, which may be the same or different, promote binding to the target (thus giving specificity and improved affinity); (2) when bound to the target, TBMs anchor the multimer structure at several positions along the scaffold, thus rigidifying the multiple chelate structure; and (3) relaxivity is enhanced to a greater extent when bound than when free in solution, thus improving imaging contrast at a specific target.
In addition to the improvement in image contrast, this invention offers synthetic advantages. A synthetically rigidified chemical framework (such as a fused ring or complex macrocycle) is not necessary since immobilization and rigidification occur upon binding by multilocus attachments to the target. Therefore, there are fewer limitations on the chemical framework structure. Additional benefits include:
a) Multilocus binding increases protein affinity and provides greater target specificity compared to a single interaction [Kramer, R. H. and Karpen, J. W., Nature, 395: pp. 710-713 (1998); Clackson, T. et al., Proc. Natl. Acad. Sci., 95: pp. 10437-10442 (1998); Rao, J. et al., Science, 280: pp. 708-711 (1998); Mann, D. A., et al., J. Am. Chem. Soc., 120: pp. 10,575-10,582 (1998); Spevak, W. et al., J. Med. Chem., 39: pp. 1018-1020.(1996); Lee, R. T. et al., Arch. Biochem. Biophys., 299: pp. 129-136 (1992)].
b) Multilocus binding slows the rate at which the agent dissociates from the target. Increasing the time that the agent remains bound results in an increased diagnostic utilization period.
c) Multilocus binding decreases the flexibility of the multiple chelate structure, reduces the local chelate motion, and thus improves the relaxivity at each metal center. Rigidification of the contrast agent in the bound state compared with the free molecule occurs only upon binding to produce greater imaging contrast. The free molecule induces a relatively small signal change compared with the bound form; consequently a surprisingly greater difference between the signal induced by the bound form relative to the signal induced by the free molecule can be attained. Contrast agents that are rigid in both the bound and unbound states lack this property.
d) The binding-dependent change in signal intensity is also applicable to other imaging modalities where a change in signal intensity may accompany binding, such as optical imaging. The signal intensity may increase or decrease upon binding. In some cases, decreased signal has been shown to correlate with the rigidity of the molecule [Rimet, O., Chauvet, M., Dell""Amico, M., Noat, G., and Bourdeaux, M. Eur. J. Biochem. (1995) 228: pp. 55-59]. In other cases, signal increases upon binding [Sudlow G., Birkett D. J., and Wade D. N. Mol. Pharmacol. 12: pp. 1052-61 (1976); Sudlow G., Birkett D. J., and Wade D. N. Mol. Pharmacol. 11: pp. 824-32 (1975); Kane C. D. and Bernlohr, D. A. Anal. Biochem. 233: pp. 197-204; Lakowica, J. R. Principles of Fluorescence Spectroscopy Plenum Press, New York, N.Y. pp. 211-213 (1983)]. Multilocus binding could provide either greatly decreased signal intensity (and therefore greatly increased signal contrast) or greatly increased intensity compared to an optical contrast agent with only a single TBM. In either case, the change in signal intensity at the target site will result in improved signal contrast as a result of contrast agent binding.
The multilocus-binding contrast agents of the invention comprise IEMs, a scaffold to which multiple IEMs are attached directly or through optional linkers, and at least two separate TBMs. The TBMs may be the same or different. In some cases, the scaffold may actually comprise the IEM or a part of the IEM, for example, some chelating moieties may also serve as the scaffold or a part of the scaffold.
These multimeric/multilocus-binding compounds are unique in that the local motion of the IEMs is restricted and bound relaxivity is greatly enhanced by non-covalent binding of at least two TBMs to the target at several separate loci along the multimer structure. These interactions allow the multimer to bind the target protein in a xe2x80x9cpseudo-cyclicxe2x80x9d or a xe2x80x9czipper-likexe2x80x9d fashion. This type of binding surprisingly decreases flexibility throughout the multimer, including the TBMS, scaffold, and individual IEMs. Thus, for IEMs that include chelates, local chelate motion is reduced and remarkably enhanced MRI signals are observed with multimers since the relaxivity is increased at each IEM. This increase distinguishes the contrast agents of the present invention from those of the prior art that bind through a single TBM and thus are not xe2x80x9cpseudo-cyclizedxe2x80x9d or xe2x80x9czipperedxe2x80x9d to the targeted site. Contrast is further enhanced with multimeric/multilocus binding structures since they also produce a relatively low signal in the unbound state.
The invention has tremendous utility for all targeted MRI and optical applications, including the targeting of image-enhancing agents to biological structures, such as serum albumin and other diagnostically relevant targets, such as blood clots, particularly in those applications where multiple binding sites for the multimeric/multilocus binding contrast agent exist. These binding sites need not be identical, just in close enough proximity to be simultaneously bound by the TBMs on the contrast agent.